Human activin A is a homodimer protein consisting of 2 polypeptide chains composed of 116 amino acids, isolated from a culture supernatant of human leukemic cell strain THP-1 (IFO 50147). Its molecular weight is about 25,000 dalton, each polypeptide contains 9 Cys residues (that is, 18 Cys residues in the dimer) and there are 9 intramolecular and intermolecular disulfide bonds (Biochemical and Biophysical Research Communications, 142, 1095-1103, 1987).
Human activin A is purified and produced by repeatedly subjecting, to multi-stage chromatography, precipitation and concentration operation, a culture supernatant obtained after stimulation of human myelocytic leukemic cell THP-1 (IFO 50147) with phorbol ester ("Saibo Kogaku" (Cell Engineering), Separate Volume 4, pp. 48-58, 1988) or a culture supernatant of recombinant CHO cells highly producing human activin A, obtained by introduction of an expression vector having human activin A cDNA (Biochemical and Biophysical Research Communications, 151, 230-235, 1988). However, there are many problems where the production process involving purification of culture supernatants as the starting material derived from animal cells is used as an industrial production process.
That is, (1) because impurities which were secreted by animal cells as the producing host or derived from fetal bovine serum etc. previously added as medium ingredients should be removed at high degrees, the yield of purified human activin A remains extremely low; (2) as compared with the case where recombinant microorganisms are used as the producing host, productivity is extremely low, and highly productive animal cells or culture apparatuses are necessary to sufficiently supply the starting material to be purified; and (3) to culture the producing host, a high concentration of fetal bovine serum should be added or a serum-free medium containing growth factors etc. should be used. However, these are extremely expensive, and there is a problem with stable availability of constant quality essential for production. As described above, there are problems with low productivity etc. in the case where human activin A is produced using animal cells, and it is necessary to solve these problems in order to establish an industrial production process.
Conventionally, various attempts have been made to solve these problems. If a microorganism such as recombinant E. coli etc. is used as the producing host, its protein productivity is generally improved 100-fold or more as compared with that of animal cells, so it can be said that replacement of animal cells by microorganisms as the producing host is an effective means of improving productivity. However, the production of human activin A by E. coli results often in denatured human activin A having different intramolecular and/or intermolecular disulfide bonds to those of the natural-type (European Patent Application Publication (EP0222491), Japanese Patent Appln. Laid-Open Publication No. 119679/88).
As used herein, denatured human activin A refers to molecules with the polypeptide chain of human activin A but having lost both its tertiary structure and biological activity, such as those with intramolecular and/or intermolecular disulfide bonds cleaved to loose the tertiary structure thus forming a monomer structure, those with the disulfide bonds transferred to form a structure different from the natural type, or those polymerized via additional intermolecular disulfide bonds.
Because such denatured human activin A has no biological activity, the molecule should be reconstituted (refolded) so as to have the same tertiary structure as that of the natural-type protein. However, it is not easy or even not possible to refold some proteins, so it is very difficult to determine their refolding conditions. Even after examination, it is not always possible to determine suitable refolding conditions particularly for certain polymeric proteins or proteins containing a large number of disulfide bonds, thus making it difficult to determine the refolding conditions.
Natural-type human activin A possesses 9 intramolecular and intermolecular disulfide bonds, so it is extremely difficult to determine the conditions for refolding of denatured human activin A and there is no prior art on the method of refolding human activin A. Meanwhile, A. J. Mason et al. examined the relationship between the efficiency of secretory expression of activin A by use of animal cells and the structure of an expression vector therefor, they reported that besides a region for the amino acid sequence of activin A, a pro-sequence region is essential for refolding and secretion of activin A, and only the region for the amino acid sequence of activin A does not cause refolding or secretion (Science, 247, 1328-1330, 1990). This report suggests that the refolding of denatured human activin A expressed by microorganisms as the producing host is very difficult.
The object of the present invention is to provide a method of refolding denatured human activin A produced by microorganisms into natural-type human activin A having a biological activity in order to constitute industrial production of human activin A.